American Society of Hematology

Figure 6.

$Figure 6. RNA targets of PMP-derived miR-24. (A) Schematic for low-throughput miRNA target identification. PMP-treated cells were lysed posttrypsinization by suspension in hyposmotic buffer followed by sonication. Whole-cell extracts were treated with RNase T1, 3′ end blocking with PNK minus lacking 3′ phosphatase activity, followed by T4 RNA ligase. RNA extracted from these samples with TRIzol was tagged with poly(dA) tails and subjected to first-strand cDNA synthesis, followed by conventional PCR with Taq polymerase using miRNA-specific 5′ primers and poly(dT) 3′ primer, direct cloning of unsorted PCR products into the pCR2.1 (TA) vector, and transformation of the DNA ligation reactions into E coli. Colonies were selected and plasmid DNA preparations were analyzed by conventional sequencing. (B) miR-24:target RNA adduct clones. TA clones with inserts matching unique sequences are shown. The insert sequences are separated in the table into the apparent miRNA segment, the cognate target/adduct segment, and the poly(A) segment. (C) mt-Nd2 and Snora75 RNA enrichment in RISC complexes following tumor cell exposure to PMPs. Shown are qPCR ratios for mt-Nd2 and Snora75 RNA content in Ago2 immunoprecipitate (IP) fractions from LLC (left) or MC-38 cells (right) treated with PMPs, relative to IP fractions from untreated cells. *P < .05 (n = 3). (D) LLC cells were transfected with 25 μg of phosphorothioate, LNA 8-nt control or antagomiR-24 (ant-miR-24), 24 hours prior to PMP exposure. RNA was isolated from cells 16 hours after exposure to PMPs or blank media. qRT-PCR was performed using 100-bp PCR fragments of each transcript, and relative expression levels were quantified using GAPDH as a housekeeping gene control, normalized to target RNA expression in untreated cells, shown as 1. *P < .001; **P < .05 (n = 7). (E) MC-38 cells treated and analyzed as in panel D. ***P < .01; #P < .02 (n = 5). All histograms, shown ± SEM. (F-G) Western blotting with α-mt-Nd2 antibodies (Nd2) of lysates of cells treated with PMPs for up to 3 days. β-actin was used as a loading control. Densitometry results are shown for Nd2/β-actin ratios, ± SEM for 3 independent experiments. *P < .05, all others n.s.$

RNA targets of PMP-derived miR-24. (A) Schematic for low-throughput miRNA target identification. PMP-treated cells were lysed posttrypsinization by suspension in hyposmotic buffer followed by sonication. Whole-cell extracts were treated with RNase T1, 3′ end blocking with PNK minus lacking 3′ phosphatase activity, followed by T4 RNA ligase. RNA extracted from these samples with TRIzol was tagged with poly(dA) tails and subjected to first-strand cDNA synthesis, followed by conventional PCR with Taq polymerase using miRNA-specific 5′ primers and poly(dT) 3′ primer, direct cloning of unsorted PCR products into the pCR2.1 (TA) vector, and transformation of the DNA ligation reactions into E coli. Colonies were selected and plasmid DNA preparations were analyzed by conventional sequencing. (B) miR-24:target RNA adduct clones. TA clones with inserts matching unique sequences are shown. The insert sequences are separated in the table into the apparent miRNA segment, the cognate target/adduct segment, and the poly(A) segment. (C) mt-Nd2 and Snora75 RNA enrichment in RISC complexes following tumor cell exposure to PMPs. Shown are qPCR ratios for mt-Nd2 and Snora75 RNA content in Ago2 immunoprecipitate (IP) fractions from LLC (left) or MC-38 cells (right) treated with PMPs, relative to IP fractions from untreated cells. *P < .05 (n = 3). (D) LLC cells were transfected with 25 μg of phosphorothioate, LNA 8-nt control or antagomiR-24 (ant-miR-24), 24 hours prior to PMP exposure. RNA was isolated from cells 16 hours after exposure to PMPs or blank media. qRT-PCR was performed using 100-bp PCR fragments of each transcript, and relative expression levels were quantified using GAPDH as a housekeeping gene control, normalized to target RNA expression in untreated cells, shown as 1. *P < .001; **P < .05 (n = 7). (E) MC-38 cells treated and analyzed as in panel D. ***P < .01; #P < .02 (n = 5). All histograms, shown ± SEM. (F-G) Western blotting with α-mt-Nd2 antibodies (Nd2) of lysates of cells treated with PMPs for up to 3 days. β-actin was used as a loading control. Densitometry results are shown for Nd2/β-actin ratios, ± SEM for 3 independent experiments. *P < .05, all others n.s.

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